Non-specific immune system enhancement

ABSTRACT

The present invention pertains to a method for non-specifically enhancing the immune system response of a mammal to an antigen which comprises the steps of (a) withdrawing leukocyte containing material from the mammal, (b) treating the withdrawn leukocytes in a manner to alter the cells, and (c) returning the treated leukocytes to the mammal. In one embodiment, the method for non-specifically enhancing the immune system response of a mammal is carried out in conjunction with artificially contacting the mammal&#39;s immune system with an antigen for a suitable period of time to stimulate the immune system. In another embodiment, the mammal&#39;s immune system is naturally exposed to the antigen prior to the non-specific enhancement of the immune system. In yet another embodiment, the invention is directed at a method for preparing an autologous non-specific leukocyte adjuvant.

This application is a continuation-in-part of U.S. Ser. No. 502,083,filed Mar. 29, 1990, now U.S. Pat. No. 5,147,289.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention pertains to methods for non-specifically enhancingthe immune system response of a subject. In one embodiment, the immuneresponse is enhanced in conjunction with the artificial contact of theimmune system of the subject with an antigen. In another embodiment, thevigor of a weak but existing immune response is enhanced. Specifically,the method comprises withdrawing leukocytes from a subject, physicallyaltering the withdrawn leukocytes, and returning the altered leukocytesto the subject to enhance the immune response.

2. Description of the Prior Art

Immune system responses may be classified as humoral responses orcell-mediated responses. A humoral response is a response which ismediated by B lymphocytes in the form of freely diffusible antibodymolecules. A cell-mediated response is a response mediated byspecifically reactive lymphocytes, such as T lymphocytes or T cells,rather than antibodies.

Basic differences exist between humoral responses and cell-mediatedresponses. The time period from exposure to an antigen until elicitationof an immune reaction is minutes to hours for a humoral response and oneor more days for a cell-mediated response. Humoral antibodies aregenerally specific for small antigenic determinants.

T lymphocytes are generally specific for antigens associated withspecific molecules on cell surfaces.

Millions of distinct T cell clones exist in a particular person. Eachclone is characterized by the proteins (the T cell receptor) carried onthe surface of the T cell. The interaction of a receptor with aparticular antigen requires that the receptor have a receptor site whichis geometrically and chemically receptive to a corresponding site on theantigen. The forces which bind a receptor to an antigen consist ofattractive forces which include hydrogen bonding, nonpolar bonding,ionic interactions, and Van der Waal's forces. The strength of theseforces is inversely proportional to the distance between the interactinggroups. Any structural variations in the geometry of the antigen caninhibit or prevent the binding of the receptor to the antigen. Once areceptor binds to an antigen on a cell, the cell may become activated,produce immunologically important molecules, divide to form manyidentical cell copies, and generally produce a strong and specificimmunological reaction.

T cell reactions or cell-mediated responses are generally a beneficialdefense of the body. A deficiency in the immune system response(immunodeficiency disease) leads to attack by pathogens, viruses,bacteria and fungi or cancer. Certain cell-mediated responses, however,are harmful because they cause tissue destruction. Examples of suchharmful immune responses are hypersensitivity reactions (delayed-typeand immediate), rejections of allografts, graft-versus-host reactions,and some allergic reactions. In addition, some autoimmune diseases arealso harmful, such as myasthenia gravis, rheumatoid arthritis, systemiclupus erythematosus, multiple sclerosis, some forms of diabetesmellitus, thyroiditis, anterior uveitis, and Grave's disease.

One such harmful cell-mediated response is the rejection of allografts.An allograft (allogeneic graft, homograft) is a graft of a cell, tissueor organ which is transferred from a donor to a recipient of the samespecies but of disparate genotype. Because of extensive polymorphism ofcertain surface glycoproteins (glycoproteins existing in different formsat different stages of development), the grafted cells almost alwayshave on their surfaces histocompatibility or transplantation antigensthat are lacking on host cells and vice versa. The host responds byrejecting the allograft through a cell-mediated response.

A graft-versus-host reaction (GVH) occurs when lymphocytes aretransferred from an immunologically competent donor (e.g., normal adult)to an allogeneic incompetent recipient (e.g., newborn). Ordinarily animmunologically competent host will destroy an allograft through acell-mediated response. However an incompetent host cannot reject such agraft and the graft instead rejects the host. These reactions haveclinical importance when normal thymus or bone marrow cells aretransferred to immunodeficient humans (e.g., infants with geneticdefects, subjects with leukemia treated with cytotoxic drugs and/orwhole-body x-irradiation).

In autoimmune diseases, the immune system fails to recognize certaincells or parts of cells as its own ("self"). Autoimmunity ischaracterized by a specific humoral or cell-mediated response againstthe constituents of the body's own tissues (autoantigens) resulting intissue destruction. This autoimmune response is characterized by theproduction of autoantibodies and autoreactive T cells.

Allergies are a hypersensitive state caused by exposure of the immunesystem to a particular allergen, and re-exposure, which causes analtered capacity to react. Allergic responses may be immediate ordelayed. In some allergic responses, the immune system responds to anormally harmless substance, such as pollen, animal dander, or dust. Inthese allergic responses, sensitized T lymphocytes react with anallergen or antigen and release lymphokines which produce inflammation.Disease results from the inflammatory reaction caused by theseenvironmental antigens. An example of such an allergic reaction isallergic contact dermatitis.

U.S. Pat. Nos. 4,321,919, 4,398,906, 4,428,744, and 4,464,166, allissued to Edelson, the contents of which are incorporated herein byreference, describe methods for specifically reducing the functioninglymphocyte population of a human subject. The Edelson referencesdescribe methods for treating the blood of a diseased subject whereinthe disease-producing blood cells have been naturally stimulated as aconsequence of the disease state. Specifically, the methods involvetreating such naturally stimulated specific human blood cells, such aslymphocytes, with a dissolved photoactivatable drug, such as a psoralen,which is capable of forming photoadducts with the DNA in the cells inthe presence of ultraviolet radiation. The lymphocytes andphotoactivatable drug are then treated extracorporeally with ultravioletradiation thereby modifying the lymphocytes. Following extracorporealirradiation, the modified specific lymphocytes are returned to thesubject. The modified specific lymphocytes are cleared from thesubject's system by natural processes, but at an accelerated pace,presumably because of disruption of cell membrane integrity, alterationof the DNA within the cells, or similar conditions, often associatedwith substantial loss of cellular effectiveness or viability.

European Patent Application publication number 284,409, published Sep.28, 1988, (U.S. Pat. No. 4,838,852), issued to Edelson et al., thecontents of which are incorporated herein by reference, describes amethod for specifically altering the immune system response of a subjectto a specific antigen. Specifically, the Edelson et al. method comprisesthe steps of (a) contacting the subject's immune system with thespecific antigen for a suitable time to artificially stimulate theimmune system, (b) withdrawing blood cell containing material, includingantigen stimulated blood cell material, from the subject, (c) treatingthe withdrawn material or cells so as to alter the specificallystimulated cells, and (d) returning the treated material or cells to thesubject. Edelson et al. states that it may be possible to alter thecells and render them incapable of recognizing an antigen by firstwithdrawing blood cell containing material from the subject, treatingthe withdrawn material to alter the cells, returning the treatedmaterial to the subject and then contacting the subject's immune systemwith a specific antigen.

European Patent Application publication no. 333,606c, published Sep. 20,1989, discloses a method for treating an autoimmune disease whichcomprises activating T lymphocyte cells specific for the autoimmunedisease by the steps of exposing the T cells to an antigen specific forthe autoimmune disease and treating the cells with a photoreactivepsoralen cross-linking agent selective for the receptor on the cellmembrane followed by photoactivation.

Specific immune unresponsiveness has been reported to be induced in vivoin rodents by immunizing the animal with autologous antigen-specificlymphoblasts obtained after in vitro sensitization, fractionation, andemulsification in Freund's complete adjuvant, L. C. Andersson et al.,Nature, 264, pp. 778-780 (1976). Use of the nonspecific adjuvant tostimulate the specific response was necessary in that study.

Pretreatment of effector cells with 8-methoxypsoralen and ultraviolet Alight has been reported to render the specific effector cells of graftrejection immunogenic for the syngeneic recipient, M. Perez et al., TheJournal of Investigative Dermatology, 92, pp. 669-676 (1989). Reinfusionof photodamaged cells results in an immunosuppressive host response thatpermits prolonged retention of histoincompatible skin grafts andspecifically inhibits in vitro and in vivo responses that correlate withallograft rejection.

Thus, a variety of methods are known to disable specific lymphocytes indiseased subjects, to attenuate specific lymphocytes to create vaccines,and to disable specific artificially-stimulated lymphocytes in healthysubjects to prevent disease. There is still a need, however, for amethod to non-specifically enhance the immune system response of asubject. Such a method would permit the stimulation of a competent orincompetent immune system prior to an anticipated natural or artificialcontact with an antigen, would permit the stimulation of an immunesystem in a subject already naturally but weakly stimulated with anantigen such as those associated with immunodeficiency disease andchronic infections, and would permit the stimulation of an immune systemin a subject about to receive a vaccine. Also, immunoregulatory T cellscapable of inhibiting undesirable immunologic reactions such as thosefound in autoimmune diseases or graft rejections could also bestimulated and weak anti-tumor responses could be enhanced. The presentinvention provides such a method for non-specifically enhancing theimmune system response of a subject optionally with or withoutartificially contacting the subject's immune system with an antigen. Thepresent invention also provides a method for preparing an autologousnon-specific leukocyte adjuvant.

SUMMARY OF THE INVENTION

The present invention pertains to a method for non-specificallyenhancing the immune system response of a mammal which comprises thesteps of (a) withdrawing leukocyte containing material from the mammal,(b) treating the withdrawn leukocytes in a manner to alter the cells,and (c) returning the treated leukocytes to the mammal. In oneembodiment, the method for non-specifically enhancing the immune systemresponse of a mammal is carried out in conjunction with the artificialcontact of the mammal's immune system with an antigen for a suitableperiod of time to stimulate the immune In another embodiment, themammal's immune system is naturally exposed to the antigen prior to thenon-specific enhancement of the immune system. In yet anotherembodiment, the invention is directed at a method for preparing anautologous non-specific leukocyte adjuvant.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention pertains to a method fornon-specifically enhancing the immune system response of a mammal whichcomprises the steps of (a) withdrawing leukocyte containing materialfrom the mammal, (b) treating the withdrawn leukocytes in a manner toalter the cells, and (c) returning the treated leukocytes to the mammal.

In another embodiment, the invention pertains to a method fornon-specifically enhancing the immune system response of a mammal to anantigen which comprises the steps of (A) enhancing the immune systemresponse by (a) withdrawing leukocyte containing material from themammal, (b) treating the withdrawn leukocytes in a manner to alter thecells, (c) returning the treated leukocytes to the mammal, and (B)artificially contacting the mammal's immune system with the antigen fora suitable period of time to stimulate the immune system. In oneexample, the method for enhancing the immune response is carried outbefore the subject's immune system is artificially contacted with theantigen. In another example, the method for enhancing the immuneresponse is carried out simultaneously with the artificial or naturalcontacting of the subject's immune system with the antigen.

In yet another embodiment, the invention pertains to a method forpreparing an autologous non-specific leukocyte adjuvant. Some antigensagainst which an immunologic reaction would be desirable are naturallyoccurring. These naturally occurring antigens include those found onmalignant cells and on undesirable disease causing T cell clones. In thecase of progressing disease, immunologic reactions against these diseasecausing cells may exist but are clearly too weak to halt the progressionof the disease. The autologous non-specific leukocyte adjuvants of thepresent invention would enhance these weak immunologic reactions andpermit disease control.

As used herein, the term "artificially stimulate" or "artificiallycontact"refers to the positive step of contacting and stimulating theimmune system of a subject with an antigen through human intervention.The term "naturally stimulate" or "naturally contact" refers to thecontacting and stimulating of the immune system of a subject with anantigen, without human intervention, as a natural consequence ofdisease. The subject (patient, host) to be treated in the presentinvention is a mammal, preferably a human.

Adjuvants are substances that when mixed with an antigen enhanceantigenicity and give a superior immune response. For example, Freund'sincomplete adjuvant is a water-in-oil emulsion which contains an antigenin an aqueous phase suspended in a light-weight paraffin oil with theaid of an emulsifying agent. On injection, this mixture induces strongpersistent antibody formation. Freund's complete adjuvant, whichcontains killed, dried mycobacteria, e.g., Mycobacterium butyricum, inthe oil phase, elicits cell-mediated immunity (delayed hypersensitivity)as well as humoral antibody formation. Because of toxicity reasons,adjuvants such as Freund's adjuvant cannot be used in humans.

Applicant has found that reinfusion of damaged nonspecific leukocytes toa subject enhances the capacity of that subject to manifest animmunologic response to specific antigens simultaneously presented tothe subject. While not wishing to be bound by theoreticalconsiderations, applicant believes that, in contrast to the reinfusionof damaged specific pathogenic leukocytes, the reinfusion of damagednonspecific nonpathogenic leukocytes to a subject does not generallyprompt an immunologic response against the nonspecific leukocytes in thesubject. Rather, such reinfusion prompts a heightened state of awarenessin the immune system which results in an general enhancement in theimmune response. Applicant believes that the damaged nonspecificnonpathogenic leukocytes act as an autologous non-specific leukocyteadjuvant.

The reinfusion of damaged pathogenic T cells, belonging to an expandedclone, leads to a specific immunological reaction against these T cellspresumably because the immune system readily responds to the largenumber of identical T cells. The reinfusion of damaged nonpathogenic Tcells, which do not belong to an expanded clone, does not lead to animmunological reaction presumably because the immune system does notrespond to smaller numbers of cells belonging to each of the very largenumber of different T cells clones. Because these damaged nonpathogenicT cells must nevertheless be cleared from the subject, the immune systemresponds by raising the level of the immune response to an excited or anenhanced level.

In the method of the present invention, leukocyte containing material iswithdrawn from a subject. Leukocytes are white blood cells which includelymphocytes, polymorphonuclear cells and monocytes. In a preferredembodiment, the leukocytes are lymphocytes which include T cells and Bcells. In a more preferred embodiment, the lymphocytes are T cells.

In a preferred embodiment, the leukocyte containing material is selectedfrom the group consisting of blood, lymph fluid, bone marrow, lymphaticorgan tissue, and any other body fluid or tissue which containsleukocytes. Lymphocytes circulate through the lymph fluid and tissuespaces and aggregate in the primary and secondary lymphatic structures,such as the thymus, spleen and lymph nodes. Hence, lymphocytes may beobtained from the lymph fluid or the primary or secondary lymphaticstructures, or even from infiltrated organ such as skin. In a morepreferred embodiment, the leukocyte containing material is blood whichcontains large amounts of lymphocytes. Withdrawal of blood andreinfusion of treated blood to a subject is simple and convenient. Suchmethods are well known in the art and are analogous to methods used inblood dialysis.

In accordance with this invention, the leukocytes withdrawn from asubject are treated to alter them, e.g., to lethally damage,functionally inactivate, or physically alter the structure of the cellmembranes or protein components. Preferably, such inactivation treatmentdisrupts the membrane integrity of the withdrawn leukocytes, and morepreferably such treatment chemically alters the cells, such as alteringthe DNA or the cytoplasmic proteins of the leukocytes, so as to makethem immunostimulatory. Such inactivation treatment may be accomplishedby several means and is within the capabilities of those skilled in theart without the need for undue experimentation. For example, leukocytesmay be inactivated by exposure of the leukocytes to ultravioletradiation in the presence of a photoactivatable agent (photopheresis),by exposure to high or low temperature, to high or low pH values, tohigh or low pressure, to hypotonic solutions, to chemotherapeutic agentssuch as mitomycin C, to ultraviolet light without a photoactivatableagent, to visible light with a photoactivatable agent such ashematoporphyrin, to monoclonal antibodies, to toxins such as ricin, orby passage of the leukocytes through resinous materials. In some cases,mere handling of the leukocytes will accomplish the desired inactivationtreatment. Preferably, the leukocytes are gently damaged by ultravioletradiation in the presence of a photoactivatable agent.

The photopheresis method of treating leukocytes involves withdrawing ofthe leukocytes, forming the withdrawn leukocytes into an extracorporealstream, flowing the stream through a treatment chamber substantiallytransparent to ultraviolet radiation, irradiating the stream in thechamber with ultraviolet radiation in the presence of thephotoactivatable agent and returning the blood to the subject. In thismethod, blood, or leukocyte cells derived from blood, are contacted witha photoactivatable agent which is dissolved in the blood by mixing thephotoactivatable agent with the blood either (a) by administering thephotoactivatable agent orally to the subject prior to withdrawing theblood, or (b) by mixing the photoactivatable agent with the bloodsubsequent to withdrawing the blood from the subject.

The photoactivatable agent may be any agent which has an affinity for animportant component of the nucleated leukocytes, such as the nucleicacid and when activated by ultraviolet radiation, is capable ofseriously injuring the cells, such as cross-linking DNA to chemicallybond the photoactivatable agent with the nucleic acid. Examples ofphotoactivatable agents are psoralens, photoactivated cortisone,photoactivated antibodies specifically reactive to malignant lymphocytesand photoactive antibodies specifically receptive to the undesirableantibodies of a subject. Other photoactivatable agents includephotoactive pyrene and monoclonal antibodies which have been linked toporphyrin molecules. A preferred class of photoactivatable agents is thepsoralens. Preferred psoralens include 8-methoxypsoralen (8-MOP) andaminomethyltrimethyl-psoralen (AMT).

The photoactivatable agent 8-methoxypsoralen occurs naturally in avariety of plants, including limes, parsley and figs and in its inactiveform (not photoactivated) is non-toxic to humans at pharmacologic doses.The photoactivatable agent 8-methoxypsoralen is a preferred agent totreat lymphocytes because it may be transformed from a biologicallyinert state by low energy ultraviolet A radiation to a transientlyexcited state capable of covalently cross-linking DNA and othermacromolecules. Ultraviolet A radiation, which passes through clearglass and some translucent plastics, activates 8-methoxypsoralen to anactive form which cross-links sister strands of DNA by formingbifunctional adducts with pyrimidine bases, thereby transforming themolecule into a potent chemotherapeutic agent. Since the half life ofthe photoactivated 8-methoxypsoralen is only in the microsecond range,tissues not simultaneously exposed to both the drug and ultraviolet Aradiation are spared the toxic effects of the active form.

Since only oral preparations of 8-methoxypsoralen are available forclinical use, the appropriate concentration of the photoactivatableagent (about 0.6 mg per kg of body weight of 8-methoxypsoralen) must beorally administered to the subject prior to the time of photopheresistreatment (about one to two hours). Blood is then withdrawn from thesubject during the photopheresis procedure. The preferred psoralenconcentration in the blood is from about 1 nanogram to about 1000micrograms per milliliter of blood. Alternatively, a known quantity ofpsoralen could be administered.

In a preferred embodiment, the flow rate of the extracorporeal bloodstream is in the range from about 10 ml/min to about 75 ml/min.Preferably, the blood stream should be irradiated with photoenergy inthe ultraviolet wavelength range (UVA, UVB, UVC). Preferably ultravioletA will be used to effectively deliver from about 0.1 joules/cm² to about100 joules/cm² to the actual leukocytes. More preferably, the radiationdose level of the blood is from about 5 joules/cm² to about 60joules/cm² to the leukocytes. The optimal ultraviolet wavelength rangefor irradiating leukocytes in the presence of 8-methoxypsoralen is fromabout 320 nm to about 400 nm, preferably from about 334 nm to about 346nm. The leukocytes may be exposed to ultraviolet radiation for a periodfrom about 5 minutes to about 6 hours. Within the optimal wavelengthrange, an approximately 270 minute exposure to ultraviolet A is requiredto provide the average leukocyte with 2 joules/cm² of leukocyte.

The photopheresis method may be carried out in a single apparatus whichcomprises a continuous centrifuge for separating leukocytes, i.e. whiteblood cells, from the blood withdrawn from the subject. The centrifugemay be used in an initial discontinuous leukapheresis step, whereinleukocytes are separated from the blood in one or more cycles. This maybe accomplished by having the subject recline in a bed and thenleukapheresing heparinized blood during 6 cycles through a continuouslyspinning centrifuge bowl, permitting removal of a total of about 240 mlof leukocyte enriched blood. This blood may then be pooled with about300 ml of plasma obtained during the same procedure from the subject(removed 2 hours following ingestion of 0.6 mg per kg of body weight of8-methoxypsoralen) and 200 ml of sterile normal saline, yielding a finalhematocrit of approximately 6.4 plus or minus 1.7% and containing fromabout 10% to about 50% of the number of lymphocytes in the subject'sblood at the initiation of the leukapheresis. The total volume may thenbe passed through a disposable sterile irradiation chamber in order toexpose it to (ultraviolet A) energy. The chamber may be in the form of asix-chambered disposable cassette.

Each chamber of the cassette may be composed of an outer polycarbonatesheath opaque to ultraviolet A and an inner ultraviolet A transparentacrylic tube surrounding a fluorescent ultraviolet A radiation source.These two walls, between which the blood is pumped, are approximately1.0 mm apart. Flow in each chamber is from bottom to top, with shuntsconnecting the top of each chamber to the bottom of the next chamber.Total volume of the cassette is abut 190 ml. Incorporated into thisultraviolet A exposure system may be an automatically reversible bloodpump to permit continuous recycling of the blood through the cassetteand temperature sensors to ensure that the blood is not heated above 41°C. Following exposure of blood to ultraviolet A, the entire volume isreturned to the subject.

The withdrawing of the leukocytes, passage of the leukocytes to thetreatment station and return of the blood to the subject may be handledin batch form but preferably is carried out as one continuous operation.In a preferred embodiment, the continuous operation comprises formingthe withdrawn material or leukocytes into an extracorporeal stream, (b)flowing the stream through a treatment chamber substantially transparentto ultraviolet radiation, and (c) irradiating the stream in the chamberwith ultraviolet radiation in the presence of the photoactivatableagent.

Methods and apparatus useful for irradiating blood are described indetail in U.S. Pat. Nos. 4,573,960, 4,568,328, 4,578,056, and 4,428,744,the contents of which are incorporated herein by reference. Additionalmethods and apparatus for irradiating blood are described in co-pending,commonly assigned U.S. patent application Ser. No. 834,292 entitled"Concurrent On-Line Irradiation Treatment Process", and U.S. patentapplication Ser. No. 834,258 entitled "Irradiation Chamber forPhotoactivation Patient Treatment system," the contents of which areincorporated herein by reference.

In a specific embodiment, the present invention provides a method forenhancing the immune system response of a mammal. In another specificembodiment, the present invention provides a method for preparing anautoiogous non-specific leukocyte adjuvant. The method comprises thefollowing steps:

(a) withdrawing leukocyte containing material from the mammal,

(b) treating the withdrawn leukocytes in a manner to alter the cells,and

(c) returning the treated leukocytes to the mammal.

In yet another specific embodiment, the present invention is directed atan autologous non-specific leukocyte adjuvant prepared by the methodwhich comprises the steps of:

(a) withdrawing leukocyte containing material from the mammal,

(b) treating the withdrawn leukocytes in a manner to alter the cells,and

(c) returning the treated leukocytes to the mammal.

In one example of the present invention, the immune response of asubject is enhanced prior to an anticipated natural contact of thesubject with an antigen. In another example, the immune response of asubject is enhanced after the subject has already been naturallycontacted with an antigen and has manifested the associated disease.

The present invention also pertains to methods for non-specificallyenhancing the immune system response of a subject in conjunction withthe artificial contacting of the subject's immune system with anantigen. In one example, the immune response is enhanced before thesubject's immune system is artificially contacted with the antigen. Inanother example, the immune response is enhanced simultaneously with theartificial contacting of the subject's immune system with the antigen.

In general, any antigen to which the immune system response is to beenhanced may be used including those antigens associated with adisorder, pathological condition or disease state. Especially usefulantigens include those associated with a disorder caused by a deficiencyof the immune system such as an immunodeficiency disease. In a preferredembodiment, the antigen is in the form of a vaccine.

The specific antigen of interest may serve to stimulate a T cellexpressing unique T cell receptors which are capable of serving asclonotypic antigens. In such a case, clonal expansion of thesecirculating aberrant T cells mediates the disorder sought to becontrolled. The present invention provides a method for enhancing aclone-specific immune reaction which limits the activity of such anaberrant population of T cells.

Exemplary immunodeficiency diseases include acquired immunodeficiencysyndrome (AIDS), certain forms of cancer, immunodeficiency of old age,and immunodeficiency following immunosuppressive therapy. Exemplaryhypersensitivity diseases include delayed-type hypersensitivityreaction, autoimmune disease, allergy, infectious disease, rejection ofallografts, and graft-versus-host reaction.

The artificial contacting of the subject's immune system with thespecific antigen may be achieved in any manner which introduces theantigen into the subject's immune system, e.g. by injection directlyinto the blood stream, the lymphatic system, the lymphoid organs, or theskin. The antigen is then permitted to be in contact with, or exposedto, the subject's immune system, for a suitable time period so as topermit stimulation of certain leukocytes specifically in response tothat antigen. This suitable period of time could be as long as one yearbut in most instances is shorter and generally is no longer than 72hours.

In a specific embodiment, the present invention provides a method forenhancing the immune system response of a mammal to an antigen. Inanother specific embodiment, the present invention provides a method forpreparing an autologous non-specific leukocyte adjuvant to an antigen.The method comprises the following steps:

(A) enhancing the immune system response by:

(a) withdrawing leukocyte containing material from the mammal,

(b) treating the withdrawn leukocytes in a manner to alter the cells,

(c) returning the treated leukocytes to the mammal, and

(B) artificially contacting the mammal's immune system with the antigenfor a suitable period of time to stimulate the immune system.

In yet another specific embodiment, the present invention provides anautologous non-specific leukocyte adjuvant to an antigen prepared by themethod which comprises the steps of:

(A) enhancing the immune system response by:

(a) withdrawing leukocyte containing material from the mammal,

(b) treating the withdrawn leukocytes in a manner to alter the cells,

(c) returning the treated leukocytes to the mammal, and

(B) artificially contacting the mammal's immune system with the antigenfor a suitable period of time to stimulate the immune system.

In one example of the present invention, the immune response of asubject is enhanced before the subject is artificially contacted with anantigen, such as when the antigen is in the form of a vaccine. Inanother example, the immune response of a subject is enhanced after thesubject has already been naturally contacted with an antigen but beforethe subject is artificially contacted with the same or a differentantigen.

When the immune system of a subject is enhanced without artificiallycontacting the immune system with an antigen, the method of the presentinvention may be used prophylactically or therapeutically. For example,the method may be used prophylactically when the immune response of asubject is enhanced prior to an anticipated natural contact of thesystem with an antigen. Hence, the competent or incompetent immunesystem of a subject may be enhanced to increase the vigor of the immunesystem response before the subject is naturally contacted with anantigen.

The method of the present invention may also be used therapeuticallysuch as when the immune response of a subject is enhanced after thesubject has already been naturally contacted by an antigen. For example,the method can be used to increase the vigor of the immune systemresponse of a subject who has chronic infections, an immunodeficiencydisease, a cancer, or an auto immune disease.

When the method of the present invention is used to enhance the immuneresponse of a subject who has an immunodeficiency disease such asacquired immunodeficiency syndrome (AIDS) or certain forms of cancer,the method may be used to invigorate an incompetent immune system. Whilethe immune system of the subject has not effectively attacked theantigen associated with the immunodeficiency disease prior to immunesystem enhancement, the method of the present invention may be used toenhance the immune response of the subject so as to induce animmunological attack on the antigen which previously was not effectivelyattacked. Similarly, the present method can be used to enhance theresponse of an incompetent immune system of a subject who is susceptibleto chronic infections.

When the method of the present invention is used to enhance the immuneresponse of a subject who has a hypersensitivity disease or autoimmunedisorder, the method may be used to correct a deficiency or imbalance inthe immune system. Hypersensitivity and autoimmune disorders arebelieved caused by aberrant T cells which are not being controlled bycorresponding suppressor cells. The method of the present invention maybe used to alter the immune response of a subject so as to prompt thesuppressor cells to mount an immunological attack on the aberrant Tcells.

When the immune system of a subject is enhanced in conjunction withartificially contacting the immune system with an antigen, the method ofthe present invention may be used prophylactically or therapeutically.For example, the method may be used prophylactically when the immuneresponse of a subject is enhanced before the subject is artificiallycontacted with an antigen. Hence, the immune system response of asubject may be enhanced to increase the vigor of the immune responseprior to administering a vaccine to a subject to improve vaccineformation.

The method of the present invention may also be used therapeuticallysuch as when the immune response of a subject is enhanced in conjunctionwith artificially contacting the immune system of a subject with anantigen, after the subject has already been naturally contacted by anantigen. Naturally stimulated subjects may also be artificiallystimulated with the same or a different antigen by the methods of thepresent invention to achieve a therapeutic result. In such a method, itmay be possible to artificially stimulate the subject according to themethods of the present invention in order to alter the immune systemresponse of the subject and achieve a beneficial therapeutic effect.

In that particular embodiment when the present invention is used toenhance the immune response of a subject who has an immunodeficiencydisease, it has been found that only those individuals who have asufficient number of helper/inducer T cells in their peripheral bloodare capable of responding to the present method. The minimum number ofnormal helper/inducer T cells required for a response to therapy is 150per cubic milliliter of blood. This number is substantially smaller thanthe approximately 1000 per cubic milliliter found in the blood of normalimmunocompetent individuals. Therefore, a further aspect of the presentinvention is to provide a method for non-specifically enhancing theimmune system response of an immunodeficient mammal to an antigen whichcomprises first determining if the mammal's helper/inducer T cell countis at least 150 per cubic milliliter of blood. If so, then leukocytecontaining material is withdrawn from the mammal, treated in a manner toalter the cells and then returned to the mammal. As used herein,"immunodeficient" means an abnormally low T cell count as a consequenceof the loss of T cells, as occurs in inflammatory bowel disease; ordestruction of T cells by cytotoxic drugs or ionizing radiation; ordisplacement of normal T cells by malignant cells, such as in multiplemyeloma or chronic lymphocytic leukemia or cutaneous T cell lymphoma; orfrom infection of T cells, as in AIDS.

Methods for determining the number of helper/inducer T cells in asubject's blood are well known in the art and include any of those thatare normally used to measure or count blood cells, such as flowcytometry methods or FACS.

The reason for the requirement of at least 150 helper/inducer T cellsper cubic milliliter of blood is that the response to the treatmentstep, such as when photopheresis is used, requires significantimmunocompetence. When diseased blood T cells are chemically altered byultraviolet activated psoralen outside the body and then returned, animmunologic reaction against these cells is induced in the patient. Ifthe number of residual normal helper/inducer T cells is too low to allowimmunocompetence, the patient cannot respond immunologically.

Throughout this application, various publications have been referenced.The disclosures in these publications are incorporated herein byreference in order to more fully describe the state of the art.

While the invention has been particularly described in terms of specificembodiments, those skilled in the art will understand in view of thepresent disclosure that numerous variations and modifications upon theinvention are now enabled, which variations and modifications are not tobe regarded as a departure from the spirit and scope of the invention.Accordingly, the invention is to be broadly construed and limited onlyby the scope and spirit of the following claims.

I claim:
 1. A method for non-specifically enhancing the immune systemresponse of an immunodeficient mammal to an antigen, the methodcomprising the steps of:a) determining if the mammal's helper/inducer Tcell count is at least 150 per cubic milliliter of blood and, if so,conducting the following steps (b)-(d); b) withdrawing leukocytecontaining material from the mammal; c) treating the withdrawnleukocytes in a manner to alter the cells; and d) returning the treatedleukocytes to the mammal.
 2. The method according to claim 1, whereinthe mammal is a human.
 3. The method according to claim 1, wherein theleukocyte containing material is selected from the group consisting ofblood, lymph fluid, bone marrow, and lymphatic organ tissue.
 4. Themethod according to claim 1, wherein the treatment of the leukocytes isby irradiating the leukocytes with ultraviolet radiation in the presenceof a photoactivatable agent.